I am having a problem with one of my drug treatments. I am performing a 72 hour treatment on a control and 3 clones of a common cell line (C2C12). My problem is that these 4 cell lines grow at different rates and my treatment further decreases cell growth (shown before).

I just finished a treatment and found that my control cell line was approaching confluency at 24 hours, so I split it and the other 3 cell lines at the same ratio (1:10) in the hope that it would be fine for the last 48 hours of the treatment. Furthermore, after the 72 hour treatment my control cell line had plenty of cells for my harvest (with treatment ~1/3 less cells), but one of the cell lines (the slowest dividing one) had a fraction of that (1/5 for the untreated and 1/10 for the treated condition). There were too few cells for me to harvest enough for any kind of reasonable extraction.

I don't think having to split the cells mid-treatment is ideal, but if I were to do it again, would you suggest I split each cell line at a different ratio to maintain equal confluency and numbers at the end of the treatment? Or are there any other solutions?

bob1 - Growth rate only matters in that I need a sufficient number of cells to assay at the end of the treatment. I think I would have to seed the cell lines at different densities (i.e. the slowest growing at a higher density and the fastest growing at a lower density). Is that valid?

No, it isn't valid to do that as there is a difference in the growth rate of most (!) cells lines depending on the density at which they are seeded. It is a classic s shape growth curve, similar to the one you would see with bacteria in liquid medium. You will probably have to go with the slowest growing cell line and work from there.

Yep, I understand the concept. So basically I'm SOL to compare these cell lines? If I seeded at the appropriate density for the slowest growing line, my fastest one will be over confluent at 72 hours - unless I only split it, but that also seems invalid.

No problem with regards to the discussion - that's what we are here for.

You may not be out of luck, if C2C12 are not contact inhibited, they should continue to grow quite rapidly while confluent. Having said that, confluency can change how cells behave, so you should check out the effect on your protein expression before starting the assay.